Nucleic Acids Research, 1981, Vol. 9, No. 12 2819-2840
© 1981
MOLECULAR BIOLOGY |
Detection of poly A+ RNA in sea urchin eggs and embryos by quantitative in situ hybridization
Department of Biology, The University of Rochester Rochester, NY 14627, USA
Received February 17, 1981. We present an improved procedure for detecting poly A tracts in situ by hybridization of 3H poly U. Glutaraldehyde fixation achieves significantly higher retention of RNA and better morphologic preservation than does Carnoy's. A dramatic increase in signal to noise is obtained by prehybridization treatment of glutaraldehyde-fixed sections with proteinase K and acetic anhydride. Measurement of the increase in poly A concentration after fertilization by solution titration and by in situ hybridization are in excellent agreement indicating that in situ measurements yield accurate relative estimates of local RNA concentrations in sections. Examination of the grain density distribution in sections of sea urchin eggs and cleaving embryos reveals no major cytoplasmic localization of poly A+ RNA, although nuclei show much less labelling and micromeres of 16-cell embryos have a small, but significant, reduction in poly A concentration.
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